CRAT Antibody

What is CRAT and what are its primary cellular functions?

CRAT (carnitine acetyltransferase) is an enzyme belonging to the carnitine/choline acetyltransferase family with specificity for short chain fatty acids. It significantly influences metabolic flux through the pyruvate dehydrogenase complex, playing a crucial role in cellular energy metabolism. Recent research has revealed CRAT's unexpected involvement in cholesterol catabolism through the bile acid synthesis pathway in cardiomyocytes, with implications for cardiac function. Additionally, CRAT depletion has been shown to promote mitochondrial DNA release into the cytosol, triggering type I interferon responses through the cGAS-STING pathway, thus linking metabolism to innate immunity . These findings demonstrate CRAT's multifunctional importance beyond its classical role in fatty acid metabolism.

What applications can CRAT antibodies be reliably used for in experimental research?

CRAT antibodies have been validated for multiple experimental applications with specific technical parameters. The polyclonal antibody 15170-1-AP has been extensively tested and published for Western Blot (WB), Immunohistochemistry (IHC), Immunoprecipitation (IP), and ELISA applications . Application-specific recommended dilutions are critical for optimal results:

It is imperative to note that these dilutions should be further titrated in each specific experimental system to achieve optimal signal-to-noise ratios .

What is the expected molecular weight of CRAT in Western blot applications?

When performing Western blot analysis, researchers should expect a discrepancy between the calculated and observed molecular weight of CRAT. While the calculated molecular weight is 71 kDa, the observed molecular weight typically ranges between 62-68 kDa on SDS-PAGE gels . This discrepancy may result from post-translational modifications, protein processing, or conformational factors. To ensure proper identification, positive controls from mouse skeletal muscle tissue, mouse heart tissue, rat skeletal muscle, or rat heart tissue – all of which show high CRAT expression – should be included in experimental designs .

What tissue-specific considerations should researchers account for when designing CRAT antibody experiments?

CRAT expression demonstrates notable tissue specificity that must be considered when designing experiments. According to validation data, CRAT antibody 15170-1-AP shows reliable positive Western blot detection in mouse skeletal muscle tissue, mouse heart tissue, rat skeletal muscle, and rat heart tissue . For immunohistochemistry applications, positive signals have been validated in mouse brain and liver tissues. When designing tissue-specific experiments, researchers should note that different antigen retrieval methods may yield varying results:

• Primary recommendation: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

This tissue-specific expression pattern aligns with CRAT's metabolic functions, particularly in tissues with high energy demands .

How can researchers optimize antigen retrieval for CRAT immunohistochemistry?

Successful CRAT immunohistochemistry depends significantly on proper antigen retrieval methods. The recommended protocol involves antigen retrieval with TE buffer at pH 9.0, which has been validated on mouse brain and liver tissues . If suboptimal results are obtained, an alternative approach using citrate buffer at pH 6.0 may be employed. Researchers should systematically compare both methods with their specific tissue samples, as certain fixation parameters and tissue types may respond differently. For formalin-fixed paraffin-embedded samples, extended retrieval times (15-20 minutes) may be necessary to fully expose the CRAT epitopes. Careful optimization of these parameters is essential for obtaining specific staining while minimizing background .

What are the most effective validation approaches for CRAT antibody specificity?

Validating CRAT antibody specificity requires a multi-faceted approach. Knockout/knockdown validation has been documented in three publications using the 15170-1-AP antibody . Researchers can validate specificity using CRAT-knockdown models in neonatal rat ventricular cardiomyocytes (NRVMs) or by comparing with CRAT-mKO (muscle-specific knockout) mice . Western blot analysis should demonstrate absence or significant reduction of the 62-68 kDa band in knockout samples compared to wild-type controls. Additionally, enzymatic activity assays provide functional validation of CRAT absence in knockout tissues. As demonstrated in published research, successful CRAT knockdown should correspond with increased acetyl-CoA levels, providing a biochemical confirmation of specificity .

How does CRAT deficiency affect cardiac function and what methods are recommended for these investigations?

CRAT deficiency has significant implications for cardiac function through multiple mechanisms. Studies employing CRAT muscle-specific knockout (CRAT-mKO) mice revealed that CRAT deletion leads to increased heart weight:body weight and heart weight:tibia length ratios, decreased ejection fraction and fractional shortening, and increased left ventricular chamber size . These phenotypes can be evaluated through:

• Echocardiography to assess functional parameters

• Gravimetric measurements to calculate heart weight ratios

• Molecular analyses to detect inflammatory markers

Interestingly, CRAT deficiency in cardiomyocytes does not directly provoke hypertrophic or profibrotic responses in vitro. Instead, it induces proinflammatory cytokine expression (IL-1β, IL-6, TNF-α) and interferon-stimulated genes . These findings suggest that CRAT's role in cardiac function is mediated through inflammatory pathways rather than direct hypertrophic signaling.

What methodological approaches can reveal the link between CRAT and innate immune responses?

Research has uncovered an unexpected connection between CRAT and innate immunity that can be investigated through several methodological approaches. RNA-sequencing analysis of CRAT-deficient cardiomyocytes reveals enrichment of interferon-stimulated genes (ISGs) and increased expression of cytoplasmic RNA/DNA sensors (Ddx58, Ifih1, Aim2) . To explore this connection, researchers should consider:

• qRT-PCR validation of ISG expression in CRAT-knockdown models

• Measurement of cytosolic mitochondrial DNA (cmtDNA) using PCR with appropriate controls

• Pharmacological interventions with cyclosporin A (CsA) to inhibit mitochondrial permeability transition pore (mPTP)

The relationship has been functionally validated through knockdown of cGAS, which abrogates ISG expression in CRAT-deficient cells, confirming the involvement of the cGAS-STING pathway . This approach demonstrates how CRAT links mitochondrial function to innate immune signaling.

What techniques are appropriate for investigating CRAT's involvement in cholesterol metabolism?

CRAT's role in cholesterol metabolism can be studied using comprehensive metabolomic analyses. Research has demonstrated that CRAT depletion promotes cholesterol catabolism through the bile acid synthesis pathway in cardiomyocytes . To investigate this connection, researchers should:

• Perform untargeted or targeted metabolomics on CRAT-deficient versus control samples

• Specifically measure 7-HOCA (7α-hydroxycholest-4-en-3-one acid) and MCA (muricholic acid) levels

• Isolate adult cardiomyocytes from CRAT-WT or CRAT-mKO mice for ex vivo metabolic studies

Research has confirmed that knockout of CRAT leads to intracellular accumulation of 7-HOCA but not MCA in adult cardiomyocytes . This methodological approach elucidates the specific point in the bile acid synthesis pathway affected by CRAT deficiency.

What computational-experimental approaches can be employed to characterize CRAT interactions with other molecules?

Researchers investigating CRAT interactions can employ combined computational-experimental approaches similar to those used for antibody-antigen studies. A generalizable methodology includes:

• High-throughput techniques to quantitatively measure binding affinities (apparent KD values)

• Site-directed mutagenesis to identify key residues in binding interactions

• Saturation transfer difference NMR (STD-NMR) to define molecular contact surfaces

• Computational modeling through automated docking and molecular dynamics simulations

For generating 3D structures of CRAT in complexes, homology models can be built using online tools like PIGS server or the knowledge-based AbPredict algorithm, which combines segments from various proteins and samples large conformational spaces to produce low-energy models . These combined approaches overcome the challenges of traditional crystallography and provide detailed structural insights.

How can researchers effectively isolate primary cardiomyocytes for CRAT functional studies?

Isolation of primary cardiomyocytes is critical for investigating CRAT function in cardiac tissue. Based on published methodologies, researchers should:

• Prepare adult cardiomyocytes from CRAT-WT or CRAT-mKO mice through Langendorff perfusion

• Confirm cardiomyocyte purity through immunostaining or flow cytometry

• Validate CRAT knockout efficiency through Western blot and enzymatic activity assays

• Process isolated cells immediately for metabolomic or immunological analyses

For neonatal rat ventricular cardiomyocytes (NRVMs), enzymatic digestion of postnatal day 1-3 rat hearts followed by pre-plating to remove fibroblasts has been successfully employed in CRAT studies . These primary cell models maintain physiological relevance and have been instrumental in delineating CRAT's role in mitochondrial homeostasis and inflammatory signaling.

What controls are essential when analyzing interferon responses in CRAT-deficient models?

When investigating interferon responses in CRAT-deficient models, rigorous controls are essential for accurate interpretation. Based on published protocols, researchers should include:

• Both positive and negative controls for CRAT knockdown/knockout verification

• Total mtDNA quantification alongside cytosolic mtDNA measurements to distinguish between changes in mitochondrial content versus mtDNA release

• Pharmacological controls using cyclosporin A (CsA) to inhibit mitochondrial permeability transition pore

• Genetic controls through simultaneous knockdown of cGAS to validate the involvement of the cGAS-STING pathway

Additionally, validation across multiple cell types (cardiomyocytes and cardiac fibroblasts) is recommended, as research has demonstrated that CRAT deficiency induces similar interferon-stimulated gene expression patterns in different cardiac cell populations .

What are the optimal storage and handling conditions for CRAT antibodies?

Proper storage and handling of CRAT antibodies are crucial for maintaining reactivity and specificity. The recommended storage conditions for antibody 15170-1-AP are:

• Store at -20°C

• Stability guaranteed for one year after shipment when properly stored

• Aliquoting is unnecessary for -20°C storage (an advantage for laboratory workflow)

• Small volume formats (20μl) contain 0.1% BSA for stability

The antibody is supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . These conditions maintain protein stability while preventing microbial contamination. When handling the antibody, researchers should minimize freeze-thaw cycles, keep the antibody on ice during experiments, and centrifuge briefly before opening to collect the full volume at the bottom of the tube.